Architectural TFs

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Odd S. Gabrielsen

Overview

DNA-binding TFs

General principles

Architectural factors

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Odd S. Gabrielsen

Recognition of response elementsActivators vrs Architectural TFs

Ordinary activators with sequence specific DNA binding Key recruitment sites for assembly of transcription complexes

Architectural transcription factors playing a more structural role in the assembly of transcription complexes

MBV4230

Odd S. Gabrielsen

Architectural TFs - brief history

Transcription activation - focus on more and more dimentions 70-ties: 1-Dimentional understanding

≥ RNAPII: TFs binding specific cis-elements required for selective transcription

TFs mediate regulatory response 80-ties: 2-Dimentional understanding

Promoters/enhancers: clusters of cis-elements complex regulation - Several buttons have to be

pushed simultaneouly Ptashnes simplification - mixed order OK

90-ties: 3-Dimentional understanding Three-dimentional assembly of TFs required for

correct biological response

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Odd S. Gabrielsen

3D protein-promoter complexes- factors dedicated architecture some factors has a pure architectural

function designated architectural transcription factors They lack a transactivation domain (TAD) Do not function out of their natural context (in contrast to ordinary

acitvators) Their function is to confer a specific 3D structure on DNA

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Odd S. Gabrielsen

Classical HMG-proteins

non-histone chromatin proteins - original defining criteria high mobility in PAGE soluble in 2-5% TCA small < 30 kDa High content of charged amino acids abundant: 1 per. 10-15 nucleosomes

MBV4230

Odd S. Gabrielsen

Classical HMG-proteins

Three classes of HMG DNA-binding proteins HMG-box family

Eks: HMG 1 and HMG 2 Bends DNA substantially Facilitators of nucleoprotein complexes

HMG-AT-hook family Eks.: HMGI(Y) Antagonizing intrinsic distortions in the conformation of AT-rich DNA

HMG-nucleosome binding family Eks.: HMG14 and 17 Mediates moderate destabilization of chromatin higher-order structure Not present in yeast or fly

HMGB

HMGA

HMGN

HMGB-proteins

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Odd S. Gabrielsen

HMG1 and 2

3 structural domains A and B with high homology (80-90 aa) acidic C-terminal

Interaction with DNA (and histones?) A and B ≈ DNA C-term ≈ histone H1 or unknown function

A+ + + +

B+ + + +

N C- - - -

DNAHiston H1?

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Odd S. Gabrielsen

HMG-boxes in architectural proteins

One or two HMG-box domains

acidicbasic

30 Asp/Glu

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Odd S. Gabrielsen

First eukaryotic architectural TF: LEF1 (Grosschedl 1992)

LEF1: a cell type-specific TF LEF1 contains an HMG-related domain LEF1: a sequence-specific TF that binds

CCTTTGAAG found in enhancer of TCR

LEF1 induces strong bending of DNA - about 130o

Induced bending brings nearly TFs in contact

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Odd S. Gabrielsen

LEF1 3D

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Odd S. Gabrielsen

LEF1 3D

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A whole family of architectural TFs with HMG-domains UBF has repeated HMG-homologous repeats

4-6 ex dimer ≈ 10 HMG-like domains activator of rRNA gener UBF-DNA complex scaffold for SL-1 recruitment

Interaction with 180 bp that is packed into a distinct structure

DNA-motif in a series of TFs: “HMG-box” designate the DNA-sequence-motif “HMG-domain” designate the protein motif

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Two subclasses of HMG-domain proteins Proteins with multiple HMG-domains

low sequence-specificity Ubiquitous - found in all cell types eks.: HMG1, HMG2, ABF-2, UBF

Proteins with single HMG-domain (moderate) sequence-specificity Cell type-specific eks.: LEF-1, SRY, TCF-1, Sox, Mat-1, Ste11, Rox1

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Characteristic DNA-binding

binds minor groove induce bending of DNA has high affinity for non-

canonical DNA-structures such as : cruciform DNA 4-way junctions cisplatin kinked DNA

+

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NMR-structures

Examples HMG1 B-domain LEF-1 SRY Yeast Nhp6p Drosophila HMG-D

Common: 3 helix L-form heliks II and III form an angle of about 80o

Conserved aromatic aa in kink Basic concave side interact with DNA

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Odd S. Gabrielsen

Similar structures of HMG domains

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Odd S. Gabrielsen

Minor groove binding, intercalation and bending

Objective: shorten the distance between cis-elements facilitating interaction between bound factors DNA <500bp relatively stiff induced bending required

Mechanism for induced bending of DNA Protein scaffold

HMG B-domain: L-shaped protein TBP: sadle

Minor groove binding DNA-binding face = hydrophobic surface that

conforms to a wide, shallow minor groove 4 residues inserted deep into the minor groove

Full or partial intercalation (“kile”)

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Intercalation in protein-induced DNA-bending Partial intercalation in the DNA helix of a protein side

chain introduces a kink in the DNA enhancing the bend Large hydrophobic residues (N-term helix I) partially intercalates between two

base pairs The A-box HMG domain has only an Ala in the X position not large enough to

intercalate,

Intercalation linked to bending also seen in other factors Partial (TBP)

Inserted side chain unstacks two basepairs side chain as stacking-partner

Full (ETS1) side chain penetrates into the helix side chain (Trp) as new stacking-partner

Result: helix axis direction altered

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Two points of intercalation, X and Y

X = major kink and intercalation site, Y=second kink due to partial intercalation

X only Y onlyX and Y

Basic tailBindsMajorgroove

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Cooperation with TFs

A major role of non-seq.spec. architectural factors is to facilitate formation of complex nucleoprotein assemblies Need interaction with sequence specific TF to be

directed to precise locations An introduced bend could facilitate binding of one

factor, and this could subsequently assist a second factor

The seq.spec. architectural factors is known to participate in the formation of complex nucleoprotein assemblies like enhanceosomes TCR and Interferon

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Odd S. Gabrielsen

Are all TFs architectural?

A large number of publications “TFx bends DNA” positive reports “TFx bends DNA” negative reports “TFx does not bend DNA”

All TFs that bind on one side of DNA will induce bending due to one-sided neutralization of charge Degree of bending will depend on ionic condition Uncertain if biologically relevant

The term “Architectural TFs“ should be reserved for factors with a particularly developed bending mechanism

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Odd S. Gabrielsen

The charge neutralization model

+++++++++++++----------

-------------------------------------------123Sp1Asymmetrical charge neutralizationBending

+++++++++++++--------------------------------------------------123Sp1--++++++++++++++Bending effect of charge neutralizationreduced in the presence of multvalent cations

2. subgruppe: HMGA

.. First described by Søren Laland, an almost forgotten discovery

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Odd S. Gabrielsen

HMGA - proteins with AT-hook

The mammalian HMGI/Y (HMGA) proteins participate in a wide variety of cellular processes including regulation of gene trx and induction of neoplastic transformation and

promotion of metastatic progression.

All members have multiple copies of a DNA-binding motif called the `AT hook' that binds to the narrow minor groove of stretches of AT-rich sequence.

The proteins have little secondary structure in solution but assume distinct conformations when bound to DNA or other proteins Their flexibility allows the HMGI/Y proteins to induce both structural changes

in chromatin substrates and the formation of stereospecific complexes called `enhanceosomes'. Reciprocal conformational changes occur in both the HMGI/Y proteins themselves and in their interacting substrates.

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Odd S. Gabrielsen

Members

4 known members Alternatively splicing gives rise to two isoform proteins, HMGA1a (HMGI)

and HMGA1b (HMGY). These two are identical in sequence except for a deletion of 11 residues between the the first and second AT hook in the latter. Alternative splicing also produces HMGA1c.

The related HMGA2 (HMGI-C) protein is coded for by a separate gene.

Conserved Homologues of the mammalian HMGA proteins have been found in yeast,

insects, plants and birds, as well as in all mammalian species examined.

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HMGA - AT-hook binding to DNA

Each HMGA protein possesses 3 similar, but independent, AT hooks which have an invariant peptide

core motif of Arg-Gly-Arg-Pro (”palindromic” consensus PRGRP) flanked on either side by other conserved positively charged residues.

The HMGA proteins bind, via the AT hooks, to the minor groove of stretches of AT-rich DNA but

recognize substrate structure, rather than nucleotide sequence.

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HMGA proteins heavily modified

The HMGA proteins are among the most highly phosphorylated proteins in the mammalian nucleus. Cell cycle-dependent phosphorylation pga cdc2 activity in the G2/M phase of the cycle. Sites: T53 and T78 situated at the N-terminal ends of the 2. and 3. AT-hook.

Phosphorylation significantly reduces (>20-fold) DNA binding. HMGA proteins are the downstream targets of a number of

signal transduction pathways that lead to phosphorylation. HMGA proteins are also acetylated

at Lys65 by CBP and at Lys71 by PCAF …as well as methylated and poly-ADP ribosylated Hypothesis: Modifications may alter DNA-binding specificity?

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Odd S. Gabrielsen

Architectural effects

Architectural effects Binding of full-length HMGA proteins can bend, straighten, unwind and induce

loop formation in linear DNA molecules in vitro.

Multiple contact points with DNA may alter conformation of DNA A single AT-hook preferentially binds to stretches of 4-6 bp of AT-rich

sequence, and partially neutralizes the negatively charged backbone phosphates on only one face of the DNA helix.

The number and spacing of AT-rich binding sites in DNA influences the conformation of bound DNA and the biological effects elicited.

HMGA may also induce conformational change in proteins HMGA forms protein-protein interactions with other transcription factors,

which alters the 3D structure of the factors resulting in enhanced DNA binding and transcriptional activation.

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Odd S. Gabrielsen

Maniatis: HMGI(Y) contributes to formation of enhanceosomes

virus-inducible enhancer in the IFN- gene (human interferon ) cis-elements for NF-kB, IRF-1, ATF-2-c-Jun

Synthetic (multiple cis-elements) enhancer ≠ natural Too high basal transcription Less induction Responds to several stimuli, while natural

enhancer only responds to virus

Biological function depends of HMGI(Y) as architectural component

HMG I(Y) First described by Lund and Laland binds AT-rich DNA in minor groove (“AT-hook”)

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Odd S. Gabrielsen

Recentverision

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Odd S. Gabrielsen

Other functions of HMGA proteins

HMGA and cancer HMGI/Y proteins are also involved in a

diverse range of other cellular processes including pathologic processes such as neoplastic transformation and metastatic progression.

Chromosomal translocations in a long 3.intron Intron 3 of the HMGA2 genes is extremely

long (>25 kb in human and >60 kb in mouse) and separates the three exons that contain the AT hook motifs from the remainds of the 3´-untranslated tail region of the gene.

Translocation within the exceptionally long third intron are commonly observed in benign mesenchymal tumors.

3. subgruppe: HMGN

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Odd S. Gabrielsen

HMGN proteins

Three functional domains of the HMGN proteins: a bipartite nuclear localization signal (NLS), a nucleosomal binding domain (NBD) and a chromatin-unfolding domain (CHUD). The CHUD domain has a net

negative charge.

Binding of HMGN proteins to nucleosomes decreases the compactness of chromatin, and facilitates trx

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Odd S. Gabrielsen

HMGN: architectural elements reducing compactness of chromatin Model of the binding of

HMGN proteins to chromatin

HMGNs interact with both the DNA and the histone component of the nucleosome The CHUD domain interacts with

the amino terminus of histone H3. May also affect H1 binding

Incorporation of HMGN proteins into chromatin is believed to reduce the compactness of the chromatin fiber.